Germ extract for cell-free protein synthesis and process for producing the same

ABSTRACT

It is intended to provide a process for producing a plant germ extract wherein the step of breaking a plant germ into fine pieces is carried out by impacting or cutting and/or in the presence of an extraction solvent, and a germ extract obtained by this process which is contaminated with little impurities unnecessary or exerting undesirable effects in cell-free protein synthesis. Thus, cell-free protein synthesis can be performed at a high stability and a high efficiency.

TECHNICAL FIELD

The present invention relates to embryo extract for cell-free proteinsynthesis and to a method for preparing the same. More specifically, theinvention relates to embryo extract for cell-free protein synthesishaving high synthesis efficiency and to a method of preparing the samein an industrially efficient manner.

BACKGROUND ART

Intracellular protein synthesis reactions proceed through the steps offirst transcribing genetic information from DNA that bears theinformation into mRNA, whereafter a ribosome translates the informationfrom this mRNA to synthesize a protein. Currently, in terms of methodsfor performing protein synthesis, which normally occurs in the cell, exvivo, such as in a test tube, a great deal of research is underway intocell-free protein synthesis wherein, for example, ribosomes areextracted from an organism and these are used to perform proteinsynthesis reactions in vitro (JP-06-098790-A, JP-06-225783-A,JP-07-000194-A, JP-09-000291-A, JP-07-147992-A). Escherichia coli, plantembryo, rabbit reticulocytes, and the like are used as sources ofribosomes for these methods.

Methods for obtaining embryo extract containing ribosomes from plantembryo for use in cell-free protein synthesis commonly consist ofgrinding plant seeds, whereafter the seed coat and the endospermfractions are removed to produce a crude embryo fraction, which isfurther washed so as to eliminate the endosperm component, whereafterthis is ground, extracted and purified.

In conventional methods for pulverizing the embryo, it was thoughtnecessary to grind the embryo as finely as possible so as to moreefficiently extract soluble components comprising protein synthesiscomponents such as ribosomes and tRNA. However, as the embryos are smalland strong, they cannot easily be ground. Accordingly, in conventionalmethods it was common to freeze the washed embryo in liquid nitrogen orthe like and then pulverize the embryo by milling or crushing with amortar, stamp mill, bowl mill, or the like. An extraction solvent wasthen added to the embryo which had been pulverized in this manner, thiswas stirred, and thereafter a solution containing embryo extractionproduct (embryo extract) was recovered and purified by centrifuging orthe like, for use as an “enzyme stock solution” for cell-free proteinsynthesis reactions. This “enzyme stock solution” contains numerousunnecessary components that are not involved in protein synthesisreactions and may also contain substances that have an inhibitory effecton protein synthesis.

Furthermore, the extract produced by the conventional methods describedabove did not contain a sufficient quantity of tRNA for the proteinsynthesis reactions, which necessitated the addition of separatelyprepared tRNA. Furthermore, it was difficult to produce large quantitiesof extract in a short period of time with the conventional methodsdescribed above.

DISCLOSURE OF THE INVENTION

In order to solve the problems described above, the present inventorsstudied methods for increasing the protein synthesis efficiency ofembryo extracts used in cell-free protein synthesis systems, formanufacturing this extract at industrially practicable levels ofefficiency, and for extracting from plant embryo the factors necessaryfor efficient protein synthesis. In this manner, the present inventionwas achieved.

That is to say, the present invention is as described hereinafter.

(1) A plant embryo extract having lowered ribonuclease activity.

(2) The embryo extract set forth above in (1), wherein the ribonucleaseactivity is no greater than 10 pg/μl, as converted to RNase A.

(3) The embryo extract set forth above in (1) or (2), wherein theribonuclease activity is lowered to no greater than 80% of theribonuclease activity of the embryo extract produced by a methodcomprising a step of mincing a plant embryo by milling or crushing inthe absence of an extracting solvent.

(4) A plant embryo extract characterized in that the DNA content thereofis no greater than 230 μg/ml, when the optical density at 260 nm (O.D.)(A₂₆₀) thereof is 90.

(5) A plant embryo extract characterized in that the total contentthereof in combined fatty acids (palmitic acid, oleic acid and linoleicacid) is no greater than 0.03 g/100 g, when the optical density at 260nm (O.D.) (A₂₆₀) thereof is 90.

(6) A plant embryo extract characterized in that the DNA content thereofis no greater than 230 μg/ml and the total combined fatty acid contentis no greater than 0.03 g/100 g when the optical density at 260 nm(O.D.) (A260) thereof is 90.

(7) The embryo extract set forth above in any one of (1) to (6), whereinthe plant embryo is the embryo of wheat, barley, rice or corn.

(8) A method for preparing plant embryo extract comprising a step ofmincing a plant embryo by impact or chopping.

(9) A method for preparing plant embryo extract comprising a step ofmincing the plant embryo in the presence of extracting solvent.

(10) The preparation method set forth above in (9) wherein theextracting solvent comprises at least one substance selected from thegroup consisting of: a buffer solution, potassium ions, magnesium ions,and a thiol antioxidant.

(11) The preparation method set forth above in (9), wherein the mincingis performed by impact or chopping.

(12) The preparation method set forth above in any one of (8) to (11),wherein the plant embryo is substantially uncontaminated by an endospermcomponent.

(13) The preparation method set forth above in any one of (8) to (12),wherein the plant embryo is the embryo of wheat, barley, rice or corn.

(14) A plant embryo extract produced by the preparation method set forthabove in any one of (8) to (13).

(15) A cell-free protein synthesis method characterized by using theembryo extract set forth above in any one of (1) to (7) or (14).

(16) A cell-free protein synthesis solution characterized by comprisingthe embryo extract set forth above in any one of (1) to (7) or (14) andATP, GTP, creatine phosphate, creatine kinase, L-amino acids, potassiumions and magnesium ions.

(17) A cell-free protein synthesis solution comprising the embryoextract set forth above in any one of (1) to (7) or (14), wherein thecell-free protein synthesis solution having sufficient protein synthesisactivity without tRNA being added.

(18) A kit for performing cell-free protein synthesis characterized bycomprising the embryo extract set forth above in any one of (1) to (7)or (14).

(19) A kit for performing cell-free protein synthesis characterized bycomprising the cell-free protein synthesis solution set forth above in(16) or (17).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the particle size distribution of groundembryo produced in Example 1.

FIG. 2 is a graph illustrating the particle size distribution of groundembryo produced in Comparative Example 1.

FIG. 3 is a graph illustrating the fluorescent intensity of GFPsynthesized in Example 2 and Comparative Example 2.

FIG. 4 is a graph illustrating the results for synthesis of DHFR inExample 7 and Comparative Example 6. The vertical axis showsradioactivity incorporated into the protein (amount of [¹⁴C]-leucineincorporated: dpm/5 μl) and the horizontal axis shows of the amount oftRNA added.

FIG. 5 is an electrophoresis profile illustrating the results ofmeasuring the ribonuclease activity of the embryo extract of the presentinvention and embryo extract produced by extraction according toconventional methods. (A) shows RNA degradation when substrate mRNA istreated with solutions containing RNase A, prepared at four differentlevels of concentration. (B) shows RNA degradation when substrate mRNAis treated with the embryo extract of the present invention, andseparately with embryo extract produced by extraction according toconventional methods. Note that M indicates a marker.

FIG. 6 is a graph showing the amount of RNA remaining when theintensities of the bands in the electrophoresis profile obtained in FIG.5 are measured, with no RNase A treatment considered as 100% (no RNAdegradation).

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention is described in further detail.

The plant embryo used in the present invention is obtained from plantseed. Examples of plant seeds that can be used normally include thoseselected from such plants of the Gramineae family as wheat, barley, riceand corn. Among these, preferred plant seeds for use in the presentinvention include the wheat, barley, rice and corn mentioned above, butwheat is particularly preferred.

As the amount of embryo contained in plant seeds is small, it isdesirable to eliminate, to as great an extent as is possible, componentsother than embryo, so as to recover the embryo efficiently. Normally,mechanical force is first applied to the plant seeds so as to produce amixture comprising embryo, crushed endosperm and crushed seed coat. Thecrushed endosperm, crushed seed coat and the like are removed from thismixture, so as to produce a crude embryo fraction (a mixture primarilycomposed of embryo but also containing crushed endosperm and crushedseed coat). It suffices that the force applied to the plant seed be of astrength sufficient to separate the embryo from the plant seed.

Normally, conventional grinding equipment is used to grind the plantseeds, so as to produce a mixture containing embryo, crushed endospermand crushed seed coat.

The plant seeds can be ground using commonly known grinding apparatusbut it is preferable to use grinding apparatus of the type that appliesimpact force to the material that is ground, such as a pin mill or ahammer mill. The degree of grinding may be suitably chosen according tothe size of the embryo of the plant seed that is used. For example,wheat grain is usually ground to a maximum length of no greater than 4mm, and is preferably ground to a maximum length of no greater than 2mm. Furthermore, it is preferable that the grinding be performed by drygrinding.

Next, a crude embryo fraction is recovered from the ground plant seedproduced, using classifier well-known per se, such as a sieve. Forexample, in the case of wheat grain, a crude embryo fraction is normallyrecovered using a mesh size of 0.5 to 2.0 mm, and preferably 0.7 to 1.4mm. Furthermore, if necessary, the seed coat, endosperm, dust and thelike contained in the crude embryo fraction produced can be removed bywind force or electrostatic force.

It is also possible to produce a crude embryo fraction by employingmethods that make use of the difference in the specific gravities ofembryo, seed coat and endosperm, such as heavy media separation. Inorder to obtain a crude embryo fraction containing a greater quantity ofembryo, a plurality of the methods described above may be combined.Furthermore, it is possible to select the embryo from the crude embryofraction produced either visually or using a color sorter, or the like.

As the endosperm component may adhere to the embryo fraction produced inthis manner, it is preferable that this be washed in order to purify theembryo. It is preferable that this be washed by dispersing/suspendingthe embryo fraction in cold water or a cold aqueous solution at atemperature that is normally no greater than 10° C. and preferably nogreater than 4° C. and washed until the washing solution is no longerclouded. It is more preferable that the embryo fraction bedispersed/suspended in an aqueous solution containing a surface activeagent, which is normally at a temperature of no more than 10° C. andpreferably at a temperature of no more than 4° C., and washed until thewashing solution is no longer clouded. It is preferable that the surfaceactive agent be nonionic, and a wide variety of surface active agentscan be used so long as these are nonionic. Specific examples of suitablesubstances include Brij, Triton, Nonidet P40, Tween, and the like, whichare polyoxyethylene derivatives. From among these, Nonidet P40 is themost suitable. These nonionic surface active agents can, for example, beused at a concentration of 0.5%.

The washing treatment may be either one of washing with water or anaqueous solution, or washing with a surface active agent. Alternatively,the two may be used together. Furthermore, this washing may be combinedwith an ultrasound treatment.

After selecting the plant embryo from the ground product, which wasproduced by grinding the plant seed as described above, the intact(capable of germinating) embryo produced by washing is minced in thepresence, or absence, of an extracting solvent and, in the latter case,this minced plant embryo undergoes extraction with an extractingsolvent, so as to produce a embryo extraction product and a solutioncontaining the embryo extraction product (hereinafter, simply, embryoextract) is separated and purified.

In the present invention, the embryo may be minced using conventionalwell-known methods such as milling or crushing as the embryo grindingmethod, but it is preferable that the embryo be minced by impact orchopping. It is particularly desirable that impact or chopping be usedwhen the embryo is minced in the absence of an extracting solvent.Herein, the expression “mince by impact or chopping” means breaking downthe plant embryo under conditions that minimize, as compared toconventional milling or crushing, the breakdown of parts of the plantembryo such as cellular membranes, cell walls, and organelles such asmitochondria, chloroplasts and the cell nucleus.

There are no particular restrictions on the apparatus and methods thatcan be used in mincing the embryo by impact or chopping, so long as theconditions described above are satisfied, but it is preferable thatdevices having a high-speed rotary blade, such as a Waring blender, beused. The speed of the rotating blade is normally no less than 1,000 rpmand preferably no less than 5,000 rpm, but is normally no greater than30,000 rpm, and preferably no greater than 25,000 rpm. The running timefor the rotating blade is normally no less than five seconds andpreferably no less than 10 seconds. There is no particular upper limiton the running time but this is normally no more than 10 minutes andpreferably no more than five minutes. There are no particularrestrictions on the temperature when the embryo is minced, so long asthe temperature does not impair the embryo's capacity for proteinsynthesis, but this temperature is preferably no greater than 10° C. andwithin a temperature range in which the mincing operation is possible,and on the order of 4° C. is particularly preferable.

Meanwhile, if the embryo is minced by milling or crushing, suchwell-known equipment as a mortar and pestle, a stamp mill, a bowl millor the like can be used to pulp the embryo by abrasion or by pressure,in the presence of an extraction solvent. In this case, solvents similarto those described above are preferred.

Examples of particularly suitable methods for mincing the embryo includemincing by impact or chopping as described above. The use of suchmethods does not completely break down cellular membranes, cell walls orsuch organelles as the cell nucleus, and the like of the embryo, butleaves at least some part thereof undestroyed. That is to say, as thecellular membranes, cell walls and organelles such as the cell nucleus,and the like, of the embryo are not broken down to a greater degree thanis necessary, it is possible to efficiently extract substances necessaryto protein synthesis, such as RNA, ribosomes and the like, which arelocalized within the cytoplasm, at high degrees of purity, withoutcontamination by impurities contained therein, such as lipids and DNA.

The embryo can be minced in the presence of an extracting solvent or inthe absence of the extracting solvent.

In cases where the embryo is minced in the presence of an extractingsolvent, the procedure can be performed as described in specific detailbelow.

By mincing the embryo (preferably by impact or chopping) in the presenceof an extracting solvent, the step of mincing the plant embryo and thestep of extracting the plant embryo, which has been minced, with anextracting solvent are carried out simultaneously. In this case, theembryo is mixed with an amount of extracting solvent sufficient forextraction, and the embryo is minced in the presence of the extractingsolvent. In terms of the amount of extracting solvent used for each gramof unwashed embryo, this is normally no less than 0.1 ml, preferably noless than 0.5 ml, and more preferably no less than 1 ml. There is noparticular upper limit on the amount of extracting solvent but this isnormally no more than 10 ml, and preferably no more than 5 ml, for eachgram of unwashed embryo. The mixture is separated by centrifuging or thelike and the supernatant produced is recovered as the embryo extract.This may be subject to a further purification step, using gel filtrationor the like.

If the mincing and the solvent extraction are performed at the same timein a single step, the embryo extract can be produced efficiently, andeven with limited breakdown of such parts of the embryos as cellularmembranes, cell walls and organelles such as the cell nucleus, it ispossible to more thoroughly extract factors necessary for proteinsynthesis, which are localized within the cytoplasm, as compared toextracting after mincing the embryo.

Furthermore, in cases where the embryo is minced in the absence of theextracting solvent, the procedure can be performed as described inspecific detail below. First, the extracting solvent is added to embryowhich has been minced, preferably by impact or chopping, withoutexcessively breaking down cellular membranes, cell walls or organellessuch as the cell nucleus, and after stirring, the mixture is separatedby centrifuging or the like, and the supernatant obtained is collectedas the embryo extract. This may be subject to a further purificationstep, using gel filtration or the like. In terms of the amount ofextracting solvent to be used and upper limits thereon, figures may beused which are similar to those in the case described above for mincingthe embryo in the presence of the extracting solvent.

In terms of the embryo which is to be minced, this may be frozen as wasconventional, or an unfrozen embryo may be used, but the use of unfrozenembryo is preferred.

An aqueous solution comprising at least one of: a buffer solution,potassium ions, magnesium ions and a thiol antioxidant, may be used asthe extracting solvent. Furthermore, calcium ions and L-amino acids maybe added as necessary. For example, solutions comprisingN-2-hydroxyethylpiperazine-N′-2′-ethanesulfonic acid (HEPES)-KOH,potassium acetate, magnesium acetate, L-amino acids and/ordithiothreitol and a solution produced by partially modifying the methodof Patterson et al. (solutions comprising HEPES-KOH, potassium acetate,magnesium acetate, calcium chloride, L-amino acids and/ordithiothreitol) can be used as the extracting solvent. The compositionsand concentrations of the various components in the extracting solventare already known per se, and compositions and concentrations commonlyused in the preparation of embryo extraction products for cell-freeprotein synthesis may be adopted.

Gel filtration may, for example, be performed using gel filtrationapparatus which has been pre-equilibrated with the solution (a solventcontaining HEPES-KOH, potassium acetate, magnesium acetate,dithiothreitol, or L-amino acids). The compositions and concentrationsof the various components in the gel filtration solution are alreadyknown per se, and compositions and concentrations commonly used in thepreparation of embryo extracts for cell-free protein synthesis may beadopted.

Following gel filtration, the embryo extract may be contaminated withmicroorganisms, and in particular, with spores such as those offilamentous bacteria (mold). It is, therefore, preferable that thesemicroorganisms be eradicated. The proliferation of microorganisms isparticularly observed in long-term (more than one day) cell-free proteinsynthesis reactions. It is, therefore, important to prevent this. Thereare no particular restrictions on the means for eradicatingmicroorganisms, but the use of antimicrobial filters is preferred. Thereare no particular restrictions on the pore size for the filter, so longas this is a size capable of eradicating microorganisms with which theembryo extract may be contaminated, but 0.1 to 1 μm is normally suitableand 0.2 to 0.5 μm is preferred. It is of note that the spore-size ofBacillus subtilis, which is in the small class, is 0.5 μm×1 μm andtherefore the use of a 0.20 micrometer filter (for example the Minisart™by Sartorius) is efficient for removing spores. When filtering, it ispreferable that a filter having a large pore size be used first,whereafter a filter having a pore size capable of eliminatingmicroorganisms by which the embryo extract may be contaminated is used.

In the present invention, it is preferable that the embryo be minced inthe presence of the extracting solvent and it is particularly preferablethat the embryo be minced by impact or chopping in the presence of theextracting solvent.

The embryo extract obtained in this manner is purified so as to besubstantially uncontaminated by endosperm comprising substances,contained or retained by the source cell itself, which inhibit proteinsynthesis function (substances that act on mRNA, tRNA, translationfactor proteins, ribosomes and the like so as to inhibit the functionthereof such as tritin, thionine, ribonuclease, phosphatase and thelike). Herein, the expression “substantially uncontaminated byendosperm” refers to embryo extracts from which endosperm componentshave been removed to an extent that ribosomes are substantially notdeadenylated. Furthermore, the expression “ribosomes are substantiallynot deadenylated” means that the ribosome deadenylation is less than 7%,and preferably 1% or less.

Furthermore, by implementing a step wherein low molecular weightsubstances that inhibit protein synthesis function, and by which thisembryo extract may be contaminated, are removed by dialysis or the like,it is possible to produce a embryo extract having even better proteinsynthesis function. Is preferable that this removal step be performed inthe presence of stabilizing components such as ATP, GTP, amino acids, orthe like.

Moreover, the embryo extract of the present invention, such as theembryo extract obtained by the procedure described above, ischaracterized by having a reduced ribonuclease activity. Herein, theterm “reduced” is intended to mean that, when compared to plant embryoextracts for cell-free protein synthesis produced by conventionalwell-known methods for preparing embryo extracts, the activity thereofis reduced, and the term specifically refers to embryo extract whereinthe ribonuclease activity, as converted to RNase A, is no greater than85%, preferably no greater than 80% and most preferably no greater than75% of the ribonuclease activity of embryo extract produced by mincingplant embryo in the absence of an extracting solvent by milling orcrushing (which is to say, by conventional methods). For example, aembryo extract having reduced ribonuclease activity of the presentinvention has a ribonuclease activity of no greater than 10 pg/μl, asconverted to RNase A.

Ribonuclease activity can be measured by methods already known per se,but specifically, using mRNA as a substrate for ribonuclease (it will beclear to those skilled in the art that there are no particularrestrictions on the type of mRNA, and that the mRNA may be freely chosenso long as the same type of mRNA is used in the comparative studies,which are based on embryo extract [embryo extract produced byconventional extraction methods] that serves as a control for the embryoextract of the present invention) and finding the degree of RNAdegradation when this mRNA is treated with the various embryo extracts,it is possible to discover the relative ribonuclease activitycontaminating the embryo extract of the present invention. Furthermore,by comparing the degree of degradation by this RNase to that of aribonuclease of known concentration, which serves as a standardpreparation, such as RNase A, it is possible to discover the amount ofribonuclease contaminating the embryo extract of the present invention.In the present specification, “converted to RNase A” means that thevalue is that would be obtained if RNase A, which is a ribonuclease of aknown concentration used as a standard preparation, were used. Thespecific procedure is described below.

Furthermore, the embryo extract of the present invention is such that,for example, when the optical density at 260 nm (O.D.) (A₂₆₀) is 90, theembryo extract obtained by the procedure described above has a DNAcontent of no more than 230 μg/ml, preferably no more than 200 μg/ml,and more preferably no more than 180 μg/ml. In the present invention,the DNA content is measured according to the following method.

Method for Measuring DNA Content

DNA content is measured using a microplate fluorophotometer (SPECTRAmaxGEMINI XS, Molecular Devices) using PicoGreen dsDNA quantitation reagent(Molecular Probes), with Calf Thymus DNA Standard (Pharmacia Biotech) asa standard sample.

First, 10 μl of proteinase K (10 mg/ml) are added to 200 μl of sampleand this is reacted overnight at 55° C. After extracting the reactionsolution in an equal volume of phenol, this is deproteinized by furtherextraction with an equal volume of phenol/chloroform (1:1) and ethanolprecipitated by adding a {fraction (1/10)} volume of 3 mol/l sodiumacetate and 2 volumes of ethanol. After washing with 70% ethanol, thisis dissolved in 10 mM/l tris hydrochloride buffer (pH 8.0) (TE)containing 1 mM/l sodium ethylenediaminetetraacetate. RNaseA (10 mg/ml)in the amount of 5 μl are added to the solution and reacted overnight at37° C., then extracted with phenol and phenol/chloroform as describedabove to remove the RNA. The sample is ethanol precipitated, washed anddissolved in 200 μl of TE. Picogreen dsDNA quantitation reagent, diluted200 fold with TE, and the sample, which has been diluted to a suitableconcentration with TE, are mixed at a 1:1 ratio, fluorescence ismeasured at 485/530 nm excitation/emission (fluorescent) wavelengths,and the DNA content is calculated using a standard curve.

Furthermore, the embryo extract of the present invention is such thatwhen the optical density at 260 nm (O.D.) (A₂₆₀) is 90, the total fattyacid content is no more than 0.03 g/100 g, preferably no more than 0.02g/100 g, and more preferably no more than 0.018 g/100 g. In the presentinvention, total fatty acid content is a value measured according to thefollowing method (gas chromatography method). In the present invention,total fatty acid content is the total content of palmitic acid, oleicacid, and linoleic acid.

Method for Measuring Total Fatty Acid Content

Acid hydrolysis was performed by adding 2 mg of heptadecanoic acid(internal standard), 4 ml of ethanol, 6.7 ml of ion exchanged water and8.3 ml of 12 mol/l hydrochloric acid to 5 ml of sample. Next, 16 ml ofethanol were added, extraction was performed by adding 100 ml of amixture of diethyl ether/petroleum ether (volume ratio 1:1), and thiswas twice extracted with 60 ml of this mixture. After washing with waterto remove the extracting solvent, saponification andmethylesterification were performed according to ACOS Official Method Ce1 b-89 (1997), whereafter measurements were performed by gaschromatography under the following operating conditions.

Gas Chromatography Operation's Conditions

-   Measurement device: Shimadzu GC-17A-   Detector: FID-   Column: J & W DB-23, φ 0.25 mm×30 mm, df. 0.25 μm-   Column temperature: 70° C. (1 minute retention)→170° C. (Temperature    rise: 10° C./min.)→210° C. (Temperature rise: 1.2° C./min)-   Inlet temperature: 250° C.-   Detector temperature: 250° C.-   Gas flow: helium 1.5 ml/minute-   Gas pressure: hydrogen 60 kPa, air 50 kPa-   Injector: splitless

The contents of the various fatty acids (palmitic acid, oleic acid andlinoleic acid) were calculated based on readings obtained according tothe following formula, so as to determine the total fatty acid content.Content in each fatty acid (g/100 g)=(E×F×H/D×G)×0.1(In the formula, D represents the peak area for heptadecanoic acid, Erepresents the peak areas for each of the fatty acids, F represents theamount of heptadecanoic acid added (mg), G represents the amount ofsample used, and H represents the sensitivity correction coefficient,which is the previously measured sensitivity of each fatty acid toheptadecanoic acid.)

Furthermore, when the embryo extract of the present invention, such asthe embryo extract obtained according to the procedure described above,is used in cell-free protein synthesis, it is possible to limit theconsumption of energy sources such as ATP, as compared to the use ofembryo extract produced by conventional extraction methods. Thissuggests that the phosphatase activity of the embryo extract of thepresent invention may be reduced.

The present invention also provides a cell-free protein synthesis methodcharacterized by using the embryo extract of the present invention.

Other than the use of the embryo extract obtained in the mannerdescribed above, the cell-free protein synthesis method of the presentinvention is performed in the same manner as conventional methods. Thismethod may be a well-known batch method, or a method wherein amino acidsand an energy source are continuously supplied, such as the continuouscell-free protein synthesis systems of Spirin et al. (A. S. Spirin etal., [1988] Science, 242, 1162-1164) and Yokoyama et al. (Kikawa et al.,The 21^(st) meeting of Molecular Biology Society of Japan, WID6) As thereaction stops when protein synthesis is performed over a long period oftime, using the batch method, the use of the latter systems in whichamino acids and an energy source are continuously provided, which allowsthe reaction to be maintained over a long period of time, make furtherincreases in efficiency possible. Furthermore, if a continuous supplysystem is used for protein synthesis, dialysis can be used. For example,large-scale continuous preparation of proteins is possible with anultrafiltration dialysis system using the embryo extract of the presentinvention as the internal dialysis solution, and a mixture containing anenergy source and amino acids as the external dialysis solution. Here,examples of the energy source include adenosine triphosphate (ATP),guanosine triphosphate (GTP), creatine phosphate, and the like, andexamples of the amino acids include the 20 types of L-amino acids.

As the embryo extract of the present invention (embryo extract producedby a method characterized in that the embryo is minced in the presenceof an extracting solvent, or embryo extract produced by a methodcharacterized in that the embryo is minced in the absence of anextraction solvent by impact or chopping and subsequently extracted withan extracting solvent) is such that cellular membranes, cell walls andsuch organelles as the cell nucleus of embryo and the like are notbroken down more than is necessary during preparation, thus greatlyreducing such as contamination by ribonuclease. Thus, as tRNA, which isa requisite for protein synthesis, is subject to little degradation byribonuclease, the embryo extract of the present invention usuallycontains an amount of tRNA that is sufficient for the protein synthesisreaction, which eliminates the necessity of adding separately preparedtRNA, as was conventional. Embryo extract of this sort functions ascell-free protein synthesis solution at least comprising a plant embryoextraction product, and in the presence of components necessary orsuitable for protein synthesis, such as ATP, GTP, creatine phosphate,creatine kinase, L-amino acids, potassium ions, magnesium ions, and thelike, it has sufficient protein synthesis activity, without tRNA beingadded. Herein, a cell-free protein synthesis solution that “hassufficient protein synthesis activity without tRNA being added” means,as described in the examples below, a cell-free protein synthesissolution which, even when tRNA is not added, has a protein synthesisactivity that is at least equivalent to the protein synthesis activityof conventional protein synthesis solutions, wherein a plant embryowhich has been frozen in liquid nitrogen or the like is milled orcrushed in a mortar or the like, and subsequently extracted so as toprepare a embryo extract to which a sufficient amount (an amount wherebythe protein synthesis reaches a plateau) of tRNA is added. Morepreferably the solution is a solution that, when no tRNA is added, has aprotein synthesis activity equal to, or greater than, the proteinsynthesis activity when tRNA is added.

Furthermore, an aqueous solution containing the embryo extract of thepresent invention, having sufficient protein synthesis activity withouttRNA being added as described above, and components necessary orsuitable for protein synthesis, such as ATP, GTP, creatine phosphate,creatine kinase, L-amino acids, potassium ions, magnesium ions, and thelike, can easily be used as a ready-made, cell-free protein synthesissolution. There are no particular restrictions on the amounts of theaforementioned components contained so long as the concentrations allowthe cell-free protein synthesis reaction to be performed. Such aready-made, cell-free protein synthesis solution allows for largeamounts of protein to be synthesized simply and efficiently, simply byadding the target translation template (mRNA), without preparing areaction solution for the protein synthesis, as was conventional. Theprotein synthesis solution can be prepared using the aforementionedextracting solvent as a solvent and adding the aforementioned componentsas necessary.

A kit for performing the cell-free protein synthesis of the presentinvention comprises at least the embryo extract or the cell-free proteinsynthesis solution described above, and may comprise optional elementssuch as other reagents and reaction vessels necessary to the cell-freeprotein synthesis, including diluents (buffer solution), dialysates,energy sources, expression vectors, expression vectors for positivecontrols, amino acids as substrate, dialysis tubes, and the like.Furthermore, the reagent kit may contain reagents used in transcriptionsystems such as RNA polymerase.

EXAMPLES

In the following, the present invention is described in further detailby way of examples, but the following examples are only intended to aidin concrete appreciation of the present invention, and the scope of thepresent invention is in no way limited to the examples described below.

Note that in the following examples: l stands for liter; ml stands formilliliter; M stands for mol/liter; mM stands for millimol/liter, and μgstands for microgram.

Example 1 Pulverization and Extraction Using a Waring Blender (1)

Hokkaido Chihoku wheat (undisinfected) was added to a mill (Pulverisette14 Rotor Speed Mill, Fritsch) at a rate of 100 g per minute, and thegrains were moderately ground at 7000 rpm. This grinding process wasrepeated four times. After recovering a fraction containing germinatableembryos with a sieve (mesh size 0.71 to 1.00 mm), the surfacing fractioncontaining the germinatable embryos was recovered by heavy mediumseparation using a mixture of carbon tetrachloride and cyclohexane(volume ratio=carbon tetrachloride:cyclohexane=2.4:1), the organicsolvent was eliminated by desiccation at room temperature, and thenimpurities such as seed coat were eliminated by air-blowing at roomtemperature to obtain a crude embryo fraction.

Next, a belt type color sorter BLM-300K (Manufacturer: AnzaiManufacturing Co., Ltd., Marketed by: Anzai Co., Ltd.) was used toselect the embryo from the crude embryo fraction by way of colordifference. This color sorter is a device comprising: means forirradiating the crude embryo fraction with light; means for detectingreflected light and/or transmitted light from the crude embryo fraction;means for comparing the detected value with a reference value; and meansfor selecting and eliminating that which is outside the standard valueor that which is within the standard value.

The crude embryo fraction was supplied onto the color sorter belt so asto produce 1,000 to 5,000 particles/m², the crude embryo fraction on thebelt was irradiated with fluorescent light and the reflected light wasdetected. The belt transport speed was 50 m/minute. A monochrome CCDline sensor (2048 pixels) was used as the photosensor.

First, in order to eliminate components darker than the embryo (seedcoat and the like), a beige colored belt was used and the standard valuewas set between the brightness of the embryo and seed coat and objectsoutside of the standard value were removed by suctioning. Next, in orderto select the endosperm, a dark green belt was used and the standardvalue was set between the brightness of the embryo and endosperm, andobjects outside of the standard value were removed by suctioning.Suctioning was performed by way of 30 suction nozzles (the suctionnozzles were aligned with one suction nozzle for each centimeter oflength) positioned approximately 1 cm above the transport belt.

By repeating this process, the embryo was selected to a embryo purity(weight ratio of embryo per gram in any sample) of no less than 98%.

Fifty grams of the wheat embryo obtained as described above weresuspended in distilled water at 4° C. and this was washed with anultrasonic cleaner until the wash solution was no longer clouded. Next,this was suspended in a 0.5% by volume solution of Nonidet P40, andwashed with an ultrasonic cleaner until the wash solution was no longerclouded, so as to produce wheat embryo from which the endosperm had beenremoved.

Next, the following operations were performed at 4° C. to produce aembryo extract (solution containing wheat embryo extraction products).First, the washed wheat embryo was placed in a Waring blender togetherwith 100 mM of extracting solvent (80 mM of HEPES-KOH [pH7.6], 200 mM ofpotassium acetate, 10 mM of magnesium acetate, 4 mM of calcium chloride,0.6 mM each of the 20 kinds of L-amino acids and 8 mM of dithiothreitol)and ground for 30 seconds at 5,000 to 20,000 rpm. Twice thereafter, theembryo which had adhered to the inner walls of the blender was scrapedoff and the grinding was repeated for 30 seconds at 5,000 to 20,000 rpm.The particle size distribution of the ground embryo produced wasmeasured with a laser-scattering granulometer (LA-920, Horiba, Ltd.).The results are shown in FIG. 1.

The resulting mixture of extract solution and ground embryo wastransferred to a centrifuge tube, centrifuged for 30 minutes at 30,000g, and the supernatant was recovered. The operation of centrifuging for30 minutes at 30,000 g and collecting the supernatant was repeated fivetimes so as to obtain an unclouded supernatant. Gel filtration wasperformed using a Sephadex G-25 column that had been pre-equilibratedwith a solution consisting of 40 mM of HEPES-KOH (pH7.6), 100 mM ofpotassium acetate, 5 mM of magnesium acetate, 0.3 mM each of the 20types of L-amino acids and 4 mM of dithiothreitol. The resultingsolution was centrifuged for 12 minutes at 30,000 g and the supernatantwas recovered to produce a solution containing wheat embryo extractionproduct. The sample concentration was adjusted with extracting solventso that the optical density at 260 nm (O.D.) (A₂₆₀) was 80 to 150(A₂₆₀/A₂₈₀=1.5).

Example 2 Preparation of a Wheat Embryo Cell-Free Protein SynthesisSystem Using Dialysis

The concentration of the solution containing wheat embryo extractionproduct produced in Example 1 was adjusted with extracting solvent sothat the optical density at 260 nm (O.D.) (A₂₆₀) was 90 and greenfluorescent protein (GFP) was synthesized according to the methoddescribed in Endo, Y. et al., PNAS, Jan. 18, 2000, Vol. 97, No. 2,559-564. The GFP activity was quantified by measuring the 510 nmfluorescent intensity at an excitation wavelength of 490 nm using aTD-360 Mini-Fluorometer by Turner Designs. As shown in FIG. 3 (indicatedas blender method in FIG. 3) the fluorescent intensity was measured tobe approximately 350,000 after 24 hours and 480,000 after 48 hours,confirming that GFP was being synthesized.

Example 3 Analysis of Solution Containing Wheat Embryo ExtractionProduct (1)

Hokkaido Chihoku wheat (undisinfected) was used to prepare a solutioncontaining wheat embryo extraction product by the same method as inExample 1. The concentration of the samples were adjusted withextracting solvent so that the optical density at 260 nm (O.D.) (A₂₆₀)was 90, and the content of DNA and RNA in each of the samples wasmeasured. The results are shown below. RNA (μg/ml) DNA (μg/ml) Sample a11411 141 Sample a2 1554 142

The methods for measuring the DNA and RNA content are as follows.

Method for Measuring DNA Content

DNA content was measured using a microplate fluorophotometer (SPECTRAmaxGEMINI XS, Molecular Devices) using PicoGreen dsDNA quantitation reagent(Molecular Probes), with Calf Thymus DNA Standard (Pharmacia Biotech) asa standard sample.

First, 10 μl of proteinase K (10 mg/ml) were added to 200 μl of sampleand this was reacted overnight at 55° C. After extracting the reactionsolution with an equal volume of phenol, this was deproteinized byfurther extraction with an equal volume of phenol/chloroform (1:1) andethanol precipitation was performed by adding a {fraction (1/10)} volumeof 3 M sodium acetate and 2 volumes of ethanol. After washing with 70%ethanol, this was dissolved in 10 mM of tris hydrochloride buffer (pH8.0) (TE) containing 1 mM sodium ethylenediaminetetraacetate. RNase A(10 mg/ml) in the amount of 5 μl was added to the solution and reactedovernight at 37° C. This was extracted with phenol and phenol/chloroformas described above to remove the RNA. The sample was ethanolprecipitated, washed and dissolved in 200 μl of TE. Picogreen dsDNAquantitation reagent, diluted 200 fold with TE, and the sample, whichhad been diluted 100 fold with TE, were mixed at a 1:1 ratio, thefluorescence was measured at 485/530 nm excitation/emission(fluorescent) wavelengths, and the DNA content was calculated using astandard curve.

Method for Measuring RNA Content

RNA content was measured using a microplate fluorophotometer (SPECTRAmaxGEMINI XS, Molecular Devices) using BioGreen RNA Quantitation reagent(Molecular Probes) as a fluorescence reagent, with Ribosomal RNAstandard (16S and 23S rRNA from E. coli) as the standard RNA.

First, 3 μl of DNase I (RNase free, 1 U/μl) were added to 100 μl of theextract solution, and this was reacted for 30 minutes at 37° C. Purifiedwater in the amount of 300 μl was added to the reaction solution andafter extracting this twice with 400 μl of water-saturated phenol, thiswas further extracted with 400 μl of chloroform to remove the DNA. Tothis were added a {fraction (1/10)} volume of 3M sodium acetate and 2volumes of ethanol, for ethanol precipitation, and this was dissolved in100 μl of TE. To 100 μl of the sample, which had been diluted 1,500 foldwith TE, was added 100 μl of 200 fold diluted BioGreen RNA Quantitationreagent and fluorescence was measured at 480/520 nm excitation/emission(fluorescent) wavelengths, and the RNA content was calculated using astandard curve.

Example 4 Analysis of the Solution Containing Wheat Embryo ExtractProduct (2)

Hokkaido Chihoku wheat (undisinfected) was used to prepare a solutioncontaining wheat embryo extraction product by the same method as inExample 1. The concentration of the samples were adjusted withextracting solvent so that the optical density at 260 nm (O.D.) (A₂₆₀)was 90, and the content of the lipids (acid hydrolysis) and total fattyacid (gas chromatography) in each of the samples was measured. Theresults are shown below. total fatty acids lipids (g/100 g) (g/100 g)Sample a3 below detection limit 0.03 Sample a4 below detection limit0.03

lipids (g/100 g) total fatty acids (g/100 g)

Sample a3 below detection limit 0.03

Sample a4 below detection limit 0.03

The method for measuring the lipid content is as follows.

Method for Measuring Lipid Content (Acid Hydrolysis)

A sample (S) in the amount of 5.0 g and 10 ml of concentratedhydrochloric acid were placed in a container, and the acid hydrolysiswas performed by heating to 80° C. in a hot water bath for 40 minutes.The digestion product produced was transferred to a Majonia tube withethyl ether and extracted by shaking with a mixed solution of diethylether/petroleum ether (volume ratio of 1:1). This was washed withion-exchanged water until the ether layer ceased to show acidity. Theether layer was recovered and transferred to a weighing vessel (W₁ g),and after removing the ether, this was further desiccated for one hourat 105° C. and weighed (W₂ g). The lipid content (g/100 g) wascalculated according to the following formula.Lipid content (g/100 g)=(W ₂ −W ₁)÷S×100Method for Measuring Total Fatty Acid Content (Gas Chromatography)

Acid hydrolysis was performed by adding 2 mg of heptadecanoic acid(internal standard), 4 ml of ethanol, 6.7 ml of ion-exchanged water and8.3 ml of 12 mol/l hydrochloric acid to 5.0 g of sample. Next, 16 ml ofethanol was added, extraction was performed by adding 100 ml of amixture of diethyl ether/petroleum ether (volume ratio 1:1), and thiswas twice extracted with 60 ml of this mixture. After washing with waterto remove the extracting solvent, saponification andmethylesterification were performed according to the ACOS OfficialMethod Ce lb-89 (1997), whereafter measurements were performed by gaschromatography under the following operating conditions.

Gas Chromatography Conditions

-   Measurement device: Shimadzu GC-17A-   Detector: FID-   Column: J & W DB-23, φ 0.25 mm×30 mm, df. 0.25 μm-   Column temperature: 70° C. (1 minute retention)→170° C. (Temperature    rise: 10° C./min)→210° C. (Temperature rise: 1.2° C./min)-   Inlet temperature: 250° C.-   Detector temperature: 250° C.-   Gas flow: helium 1.5 ml/minute-   Gas pressure: hydrogen 60 kPa, air 50 kPa-   Injector: splitless

The contents of the various fatty acids (palmitic acid, oleic acid andlinoleic acid) were calculated based on the readings obtained accordingto the following formula, so as to determine the total fatty acidcontent.Content in each fatty acid (g/100 g)=(E×F×H/D×G)×0.1(In the formula, D represents the peak area for the heptadecanoic acid,E represents the peak area of each of the fatty acids, F represents theamount of heptadecanoic acid added (mg), G represents the amount ofsample used and H represents the sensitivity correction coefficient,which is the previously measured sensitivity of each fatty acid toheptadecanoic acid.)

Example 5 Analysis of Solution Containing Wheat Embryo ExtractionProduct (3)

Okayama Shirasagi wheat (undisinfected) was used to prepare a solutioncontaining wheat embryo extraction product by the same method as inExample 1, the concentration of the samples were adjusted withextraction buffer solution so that the optical density at 260 nm (O.D.)(A₂₆₀) was 90, and the content of DNA and RNA was measured by the samemethod as in Example 3.

The results are shown below. RNA (μg/ml) DNA (μg/ml) Sample a5 1401 123

Comparative Example 1 Frozen Pulverization Extraction Using a Mortar (1)

Using the same method as in Example 1, Hokkaido Chihoku wheat(undisinfected) was ground and separated to produce a crude embryofraction and the embryo was selected to a embryo purity of no less than98% using the color sorter. Furthermore, the embryo was washed and theendosperm removed by the same method as in Example 1 to produce wheatembryo.

Next, the following operations were performed at 4° C. to produce asolution containing wheat embryo extraction product. First, the washedwheat embryo was frozen in liquid nitrogen and pulverized in a mortar.The particle size distribution of the resulting ground embryo is shownin FIG. 2.

The extracting solvent (80 mM of HEPES-KOH (pH 7.6), 200 mM of potassiumacetate, 10 mM of magnesium acetate, 4 mM of calcium chloride, 0.6 mM ofeach of the 20 types of L-amino acids and 8 mM of dithiothreitol) in theamount of 10 ml were added to the mortar and mixed. The resultingmixture of extract solution and ground embryo was transferred to acentrifuge tube, centrifuged for 30 minutes at 30,000 g, and thesupernatant was recovered. This was further centrifuged under the sameconditions as in Example 1 to produce an unclouded supernatant, andafter gel filtration, this was centrifuged to produce a supernatant,which was a solution containing wheat embryo extraction product. Thesample concentration was adjusted with extracting solvent so that theoptical density at 260 nm (O.D.) (A₂₆₀) was 170 to 250 (A₂₆₀/A₂₈₀=1.5).

Comparative Example 2 Preparation of Dialysis-Based Wheat EmbryoCell-Free Proteins Synthesis System

The concentration of the solution containing wheat embryo extractionproduct obtained in Example 1 was adjusted with extracting solvent sothat the optical density at 260 nm (O.D.) (A₂₆₀) was 90, and Greenfluorescent protein (GFP) was synthesized according to the methoddescribed in Endo, Y. et al., PNAS, Jan. 18, 2000, Vol. 97, No. 2,559-564. The GFP activity was quantified by measuring the 510 nmfluorescent intensity at an excitation wavelength of 490 nm using aTD-360 Mini-Fluorometer by Turner Designs. As shown in FIG. 3 (indicatedas mortar method in FIG. 3), the fluorescent intensity was observed tobe approximately 200,000 after 24 hours, and approximately 260,000 after48 hours, confirming that GFP was being synthesized, but the values werelower than those in Example 2.

Comparative Example 3 Analysis of Solution Containing Wheat EmbryoExtraction Product (1′)

Hokkaido Chihoku wheat (undisinfected) was used to prepare a solutioncontaining wheat embryo extraction product by the same method as inComparative Example 1. The concentration of the samples were adjustedwith extraction solvent so that the optical density at 260 nm (O.D.)(A₂₆₀) was 90, and the content of DNA and RNA in each of the samples wasmeasured by the same method as in Example 3. The results are shownbelow. RNA (μg/ml) DNA (μg/ml) Sample b1 1567 283 Sample b2 1574 283Sample b3 1578 232

As is made clear from the results above, the RNA content wasapproximately equal to the value for the solution containing embryoextraction product obtained in Example 3 (the ratio of sample a1 tosample b1 being approximately 1.1 times) and the DNA content wasapproximately double.

Comparative Example 4 Analysis of Solution Containing Wheat EmbryoExtraction Product (2′)

Hokkaido Chihoku wheat (undisinfected) was used to prepare a solutioncontaining wheat embryo extraction product by the same method as inComparative Example 1. The concentration of the samples were adjustedwith extracting solvent so that the optical density at 260 nm (O.D.)(A₂₆₀) was 90, the lipid (acid hydrolysis) and fatty acid (gaschromatography) content in each of the samples was measured by the samemethod as in Example 4. The results are shown below. total fatty acidslipids (g/100 g) (g/100 g) Sample b4 0.1 0.06 Sample b5 0.05 0.05

lipids (g/100 g) total fatty acids (g/100 g)

Sample b4 0.1 0.06

Sample b5 0.05 0.05

As is made clear from the results above, the lipid and total fatty acidcontents were both higher than the content values for the solutioncontaining embryo extraction product obtained in Example 4. The totalfatty acid content was double, when comparing sample a3 and sample b4.

Example 6 Pulverization Extraction Using a Waring Blender (2)

Hokkaido Chihoku wheat (undisinfected) was used and a solutioncontaining wheat embryo extraction product was produced by the samemethod as in Example 1, other than the fact that an extracting solventthat did not contain calcium chloride (80 mM of HEPES-KOH[pH7.8], 200 mMof potassium acetate, 10 mM of magnesium acetate, 0.6 mM each of the 20types of L-amino acids and 8 mM of dithiothreitol) was used as theembryo extracting solvent. The sample concentration was adjusted withextracting solvent so that the optical density at 260 nm (O.D.) (A₂₆₀)was 80 to 150 (A₂₆₀/A₂₈₀=1.5).

Example 7 Wheat Embryo Cell-Free Protein Synthesis Reaction Using theBatch Method (1)

The concentration of the solution containing wheat embryo extractionproduct obtained in Example 6 was adjusted with extracting solvent sothat the optical density at 260 nm (O.D.) (A₂₆₀) was 110 and thereaction solution was prepared, and the reaction of itself wasperformed, according to a known method (Madin, K. et al., Proc. Natl.Acad. Sci. USA, 2000, 97, 559-564) which was partially modified. That isto say, a reaction solution having the composition shown below in (*1)was prepared.

DHFR mRNA (with no cap structure) prepared by transcribing adihydrofolate dehydrogenase (DHFR) gene incorporated in an expressionvector (pEU), which is specifically intended for use in wheat embryocell-free protein synthesis systems, was used as a model translationtemplate (WO 01/27260), 60 μg/ml were added and the synthesis reactionwas performed at 26° C. The synthesis activity was measured based on the[¹⁴C]-leucine incorporated into the protein in 5 μl of reaction solutionafter two hours of reaction at 26° C.

Here, in order to determine whether or not addition of tRNA wasnecessary, synthesis was performed according to four different caseswherein 0 μg/ml, 50 μg/ml, 100 μg/m, and 150 μg/ml of tRNA were added,as a result of which it was understood that sufficient synthesisactivity was achieved even without the addition of tRNA (indicated asblender method in FIG. 4). Furthermore, these results also made it clearthat it was possible to produce a solution containing embryo extractionproduct having sufficient protein synthesis activity, even when therewas no calcium in the extraction solvent.

(*1) Composition of the Solutions for Wheat Embryo Cell-Free ProteinSynthesis

The reaction solutions comprise 24% by volume of wheat embryo extracthaving an optical density of 200 at 260 nm (O.D.) (A₂₆₀) (and thus,43.5% with an optical density of 110 at 260 nm [O.D.] [A₂₆₀]) and hasthe following component composition. 30 mM of HEPES-KOH (pH 7.6), 95 mMof potassium acetate, 2.65 mM of magnesium acetate, 2.85 mM ofdithiothreitol, 1.2 mM of adenosine triphosphate (ATP), 0.25 mM ofguanosine triphosphate (GTP), 16 mM of creatine phosphate, 0.5 mg/ml ofcreatine kinase, 0.380 mM of spermidine, 20 kinds of L-amino acids (0.3mM each), 1,000 units/ml of ribonuclease inhibitor (RNase inhibitor).

Comparative Example 5 Frozen Pulverization Extraction With a Mortar (2)

Hokkaido Chihoku wheat (undisinfected) was used and a solutioncontaining wheat embryo extraction product was produced by the samemethod as in Example 1, other than the fact that the extracting solventthat did not contain calcium chloride (80 mM of HEPES-KOH (pH7.8), 200mM of potassium acetate, 10 mM of magnesium acetate, 0.6 mM of each ofthe 20 types of L-amino acids, and 8 mM of dithiothreitol) was used asthe embryo extracting solvent. The sample concentrations were adjustedwith extracting solvent so that the optical density at 260 nm (O.D.)(A₂₆₀) was 170 to 250 (A₂₆₀/A₂₈₀=1.5).

Comparative Example 6 Wheat Embryo Cell-Free Protein Synthesis ReactionUsing the Batch Method

The concentration of the solution containing wheat embryo extractionproduct obtained in Comparative Example 5 was adjusted so that theoptical density at 260 nm (O.D.) (A₂₆₀) was 210, the reaction solutionwas prepared in the same manner as in Example 7, protein wassynthesized, and the [¹⁴C]-leucine that was incorporated into theprotein was measured. Note that reaction solutions were used comprising24% by volume of wheat embryo extract that had an optical density of 200at 260 nm (O.D.) (A₂₆₀) (and thus, 22.8% with an optical density of 210at 260 nm [O.D.] [A₂₆₀]).

As is made clear by FIG. 4, as in Example 7, synthesis was performedaccording to four different cases wherein 0 μg/ml, 50 μg/ml, 100 μg/m,and 150 μg/ml of tRNA were added, as a result of which it was understoodthat sufficient synthesis activity was not achieved when tRNA was notadded (indicated as mortar method in FIG. 4).

Example 8 Measurement of Ribonuclease Activity

(1) Embryo Extract

The embryo extract of the present invention (Example 1) and extractproduced according to conventional methods (Comparative Example 1) wereused as the wheat embryo extracts for measurement of ribonucleaseactivity. In order to measure the activity, a conventional embryoextract was used, which contained 30 mM of Hepes-KOH (pH 7.8), 1.2 mM ofATP, 0.25 mM of GTP, 16 mM of creatine phosphate, 2 mM ofdithiothreitol, 0.3 mM spermidine, 0.3 mM of the 20 kinds of aminoacids, 2.5 mM of magnesium acetate, 100 mM of potassium acetate, 0.005%of sodium azide (hereinafter referred to as D.B.) and which was appliedto a MicroSpin G-25 Column. The extract of the present inventioncontained D.B. The concentrations of the two embryo extracts (the embryoextract of the present invention and the embryo extract produced byconventional extraction) were made equal based on OD₂₆₀ measurementvalues.

(2) Preparation of the Reaction Solution

pEU-DHFR (see WO 01/27260) was subject to PCR amplification using asense primer and an antisense primer and this was used as a template intranscription that produced mRNA, which was used as the substrate forthe ribonuclease. The transcription was performed with a 400 μl reactionsystem, which was prepared so as to contain 80 mM of Hepes-KOH, 16 mM ofmagnesium acetate, 2 mM of spermidine, 10 mM of DTT, 3 mM of NTP, 1 U/μlof SP6RNA polymerase, 1 U/μl of RNase inhibitor (RNasin) and 10% PCRproduct, and which was incubated for three hours at 37° C. In caseswhere the mRNA was labeled with radioisotope ³²P, 8 μl of [α-³²P] UTPwas added to a 400 μl reaction system having a UTP concentration of 1.2mM. After incubation, the solution was centrifuged for 2 minutes at10,000 g, 53 μl of 7.5 M ammonium acetate and 1 ml of ethanol were addedto the supernatant, and this was centrifuged for 15 minutes at 20,000 g.The precipitate was washed with 1 ml of 70% ethanol, whereafter RNAdissolved in 80 μl of D.B. was used. The various compositions are asshown below. mRNA (no radioisotope) 5 × TB  80 μl 25 mM of NTP  48 μlSP6 RNA polymerase  5 μl RNase inhibitor  5 μl DNA  40 μl Milli-Q water222 μl 400 μl

mRNA (with radioisotope) 5 × TB  80 μl 25 mM of ATP CTP GTP  48 μl 10 mMof UTP  8 μl [α-³²P] UTP SP6 RNA polymerase  5 μl RNase inhibitor  5 μlDNA  40 μl Milli-Q water 214 μl 400 μl(3) RNA Degradation Control (Assay Using RNase A as a StandardPreparation)

As an RNA degradation control, variously, 30 pg, 300 pg, 3 ng and 30 ngof RNase A were added to 2 μl of mRNA (no radioisotope) and 1 μl of mRNA(with radioisotope) and the reaction systems were adjusted to 30 μl withD.B. and incubated for 30 minutes at 26° C. The various compositions areas shown below. mRNA (no radioisotope)  2 μl  2 μl  2 μl  2 μl  2 μlmRNA (with radioisotope)  1 μl  1 μl  1 μl  1 μl  1 μl RNase A D. B. —30 pg 300 pg  3 ng 30 ng Total 30 μl 30 μl  30 μl 30 μl 30 μl

For ribonuclease activity in the embryo extract of the present inventionand the embryo extract resulting from conventional methods, 2 μl of mRNA(no radioisotope) and 1 μl of mRNA (with radioisotope) were added to thereaction systems, together with 8 μl or 12 μl of the extracts, so thatthe final densities were 40 OD₂₆₀/ml, and these were adjusted to 30 μlwith D.B. Four tubes of each were prepared and incubated at 26° C.Embryo Embryo extract of extract resulting the present from conventionalinvention methods mRNA  2μl  2μ (no radioisotope) mRNA  1μ  1μ (withradioisotope) embryo extract  8μ 12μ D. B. 19μ 15μ Total 30μ 30μ

After incubation for 10 minutes and 30 minutes, these were frozen withliquid nitrogen to stop the reaction. Milli-Q water in the amount of 255μl, 15 μl of 10% SDS, and 300 μl of phenol/Milli-Q water were added, andthis was vortexed for 10 minutes, and centrifuged for 10 minutes at20,000 g. An amount of 300 μl of the aqueous phase was transferred to anew tube, 300 μl of phenol/Milli-Q was added, and this was vortexed for10 minutes, and centrifuged for 10 minutes at 20,000 g. An amount of 300μl of the aqueous phase was transferred to a new tube, 300 μl ofchloroform was added, and this was vortexed for 10 minutes, andcentrifuged for 1 minute at 20,000 g. An amount of 300 μl of the aqueousphase was transferred to a new tube, 30 μl of 5 M sodium chloride and750 μl of ethanol were added, and this was centrifuged for 15 minutes at20,000 g. The precipitate was washed in 1 ml of 70% ethanol anddissolved in 54 μl of Milli-Q water. An amount of 6 μl of 10× Samplebuffer (0.25% Bromophenol blue, 50 mM of Tris-HCl pH7.6, 6% glycerol)was added and a 5 μl sample was electrophoresed on 1.2% agarose. Thepresence or absence of RNA degradation was verified by way of anautoradiogram (FUJIFILM: BAS-2500). The results are shown in FIG. 5.Note that M indicates a marker. (A) is a graph showing the RNAdegradation when an mRNA (substrate) is treated with a solutioncontaining RNase A, prepared at four levels of concentration. (B) is agraph showing the RNA degradation when an mRNA (substrate) is treatedwith the embryo extract of the present invention, and separately withembryo extract produced by extraction according to conventional methods.

In RNase A treatment, the mRNA was almost entirely degraded by 100 pg/mlof RNase A. The band intensities for the embryo extract of the presentinvention and the conventional embryo extract were calculated based onthe PLS using ImageGauge (FUJIFILM: BAS2500), and taking the RNase Auntreated sample as 100%. The results are shown in FIG. 6. It wasunderstood that the ribonuclease content of the extract of the presentinvention was no greater than 10 pg/μl as converted to RNase A.

It is not absolutely clear why a embryo extract having the excellentcharacteristics described above can be produced by means of the presentinvention but the reasons may be as follows.

A system for protein synthesis in a plant embryo (ribosomes, tRNA,aminoacyl-tRNA synthetases, translation factors, and the like) isprepared for active protein synthesis, which begins in accordance withthe supply of mRNA, which migrates to the cytoplasm after beingtranscribed in the cell nucleus during germination, but all of thesefactors are localized in cytoplasm alone, where the translation reactiontakes place. In other words, the factors that contribute to proteinsynthesis are not found in components other than the cytoplasm (cellularmembranes, cell walls, organelles such as the cell nucleus, or the like,of the embryo). Cellular membranes, cell walls, organelles such as thecell nucleus, and the like contain unnecessary DNA, lipids and the like,which do not contribute to protein synthesis. Thus, solutions containingembryo extraction product produced by conventional methods, whereinfrozen embryo was pulverized by milling or crushing with a mortar, stampmill, bowl mill or the like, and thereafter extracted with an extractingsolvent, contain large quantities of these unnecessary components whichdo not contribute to the protein synthesis reaction.

Components which do not contribute to the protein synthesis reactioninclude those which inhibit the protein synthesis reaction and thosehaving a disruptive effect on Higher-Order Structure Formation andfunctional analysis of synthesized products. For example, DNA is anacidic polymer that binds to basic proteins such as ribosome proteinsand inhibits the translation process. It is also possible that duringsynthesis of DNA binding proteins, some embryo-derived DNA fragmentshave base sequences that bind strongly thereto. Furthermore, lipidcomponents often interfere with the protein purification process and itis generally necessary to eliminate them in a preprocessing operation.In addition, it is possible that some low molecular weight substanceshave an inhibitory effect on protein synthesis.

Furthermore, contamination of ribonuclease results in degradation oftemplate mRNA, while contamination of phosphatase results in degradationof energy sources such as ATP.

The present invention provides a embryo extract (which is useful incell-free protein synthesis systems) wherein the embryo is minced in thepresence of the extracting solvent, or wherein the embryo is minced byimpact or chopping whereafter extraction is performed by addingextracting solvent. By employing the novel technique of mincing embryoby impact or chopping, which was not conventionally used, cellularmembranes, cell walls, organelles such as the cell nucleus, and thelike, are not broken down more than is necessary, whereby it is possibleto avoid the conventional method's problem of extracting components thatwere not necessary to, or which had a negative impact on, the proteinsynthesis reaction. Furthermore, even in cases where the embryo extractis prepared employing such conventional methods as mincing the embryo bymilling or crushing, by performing this mincing in the presence of theextracting solvent, extraction can be performed more rapidly, and as aconsequence, cellular membranes, cell walls, organelles such as the cellnucleus, and the like, are not broken down more than is necessary,whereby it is again possible to avoid the conventional method's problemof extracting components that were not necessary to, or which had anegative impact on, the protein synthesis reaction.

Possibilities for Industrial Use

By virtue of the present invention, it is possible to efficientlyextract factors necessary for protein synthesis from the cytoplasm andproduce a embryo extract low in impurities such as DNA and lipids, whichare contained in cellular membranes, cell walls, organelles such as thecell nucleus, and the like, allowing for stable provision of a embryoextract for high-efficiency cell-free protein synthesis and a cell-freeprotein synthesis solution. The use of the embryo extract for proteinsynthesis, or the solution for protein synthesis, of the presentinvention makes it possible to stably achieve high-efficiency cell-freeprotein synthesis. Furthermore, as it is possible to prepare largequantities of high purity embryo extract in a short period of time, thisis extremely useful in large-scale production of cell-free proteinsystems, such as the large-scale production of novel enzymes andantibodies in the field of evolutionary molecular engineering.

The present application is based on Japanese patent application2002-23138, Japanese patent application 2002-23139, and Japanese patentapplication 2002-23140, the contents of which are incorporated into thepresent specification in their entireties.

1. A plant embryo extract having lowered ribonuclease activity.
 2. Theembryo extract according to claim 1, wherein the ribonuclease activityis no greater than 10 pg/μl, as converted to RNase A.
 3. The embryoextract according to claim 1, wherein the ribonuclease activity islowered to no greater than 80% of the ribonuclease activity of theembryo extract produced by a method comprising a step of mincing a plantembryo by milling or crushing in the absence of an extracting solvent.4. A plant embryo extract characterized in that the DNA content thereofis no greater than 230 μg/ml, when the optical density at 260 nm (O.D.)(A₂₆₀) thereof is
 90. 5. A plant embryo extract characterized in thatthe total content thereof in combined fatty acids (palmitic acid, oleicacid and linoleic acid) is no greater than 0.03 g/100 g, when theoptical density at 260 nm (O.D.) (A₂₆₀) thereof is
 90. 6. A plant embryoextract characterized in that the DNA content thereof is no greater than230 μg/ml and the total combined fatty acid content is no greater than0.03 g/100 g when the optical density at 260 nm (O.D.) (A260) thereof is90.
 7. The embryo extract according to claim 1, wherein the plant embryois the embryo of wheat, barley, rice or corn.
 8. A method for preparingplant embryo extract comprising a step of mincing a plant embryo byimpact or chopping.
 9. A method for preparing plant embryo extractcomprising a step of mincing the plant embryo in the presence ofextracting solvent.
 10. The preparation method according to claim 9wherein the extracting solvent comprises at least one substance selectedfrom the group consisting of: a buffer solution, potassium ions,magnesium ions, and a thiol antioxidant.
 11. The preparation methodaccording to claim 9, wherein the mincing is performed by impact orchopping.
 12. The preparation method according to claim 8, wherein theplant embryo is substantially uncontaminated by an endosperm component.13. The preparation method according to claim 8, wherein the plantembryo is the embryo of wheat, barley, rice or corn.
 14. A plant embryoextract produced by the preparation method according to claim
 8. 15. Acell-free protein synthesis method characterized by using the embryoextract according to claim
 1. 16. A cell-free protein synthesis solutioncharacterized by comprising the embryo extract according to claim 1 andATP, GTP, creatine phosphate creatine kinase, L-amino acids, potassiumions and magnesium ions.
 17. A cell-free protein synthesis solutioncomprising the embryo extract according to claim 1, wherein thecell-free protein synthesis solution having sufficient protein synthesisactivity without tRNA being added.
 18. A kit for performing cell-freeprotein synthesis characterized by comprising the embryo extractaccording to claim
 1. 19. A kit for performing cell-free proteinsynthesis characterized by comprising the cell-free protein synthesissolution according to claim
 16. 20. The embryo extract according toclaim 4, wherein the plant embryo is the embryo of wheat, barley, riceor corn.
 21. The preparation method for preparing plant embryo extractaccording to claim 4, comprising a step of mincing the plant embryo byimpact or chopping.
 22. The preparation method for preparing plantembryo extract according to claim 4, comprising a step of mincing theplant embryo in the presence of an extracting solvent.
 23. Thepreparation method according to claim 22 wherein the extracting solventcomprises at least one substance selected from the group consisting of:a buffer solution, potassium ions, magnesium ions, and a thiolantioxidant.
 24. The preparation method according to claim 22, whereinthe mincing is performed by impact or chopping.
 25. The preparationmethod according to claim 21, wherein the plant embryo is substantiallyuncontaminated by an endosperm component.
 26. The preparation methodaccording to claim 21, wherein the plant embryo is the embryo of wheat,barley, rice or corn.
 27. A plant embryo extract produced by thepreparation method according to claim
 21. 28. A cell-free proteinsynthesis method characterized by using the embryo extract according toclaim
 20. 29. A cell-free protein synthesis solution characterized bycomprising the embryo extract according to claim 20, ATP, GTP, creatinephosphate, creatine kinase, L-amino acids, potassium ions and magnesiumions.
 30. A cell-free protein synthesis solution comprising the embryoextract according to claim 20, wherein the cell-free protein synthesissolution having sufficient protein synthesis activity without tRNA beingadded.
 31. A kit for performing cell-free protein synthesischaracterized by comprising the embryo extract according to claim 20.32. A kit for performing cell-free protein synthesis characterized bycomprising the cell-free protein synthesis solution according to claim30 or 31.